The Research Of The Relationgship Between Candida Albicans ERG11 Gene Mutation And Its Drug Resistance

BackgroundWith the wide use of antibiotics, immunosuppressive agents, corticosteroids, antitumor drugs, and wide using of invasive operation, such as breathing machine, trachea cannula, pulmonary fungal infection is riseing rapidly. The identification of fungus is a difficult problem in clinical microbiology laboratory; it has a very important influence for the early clinical diagnosis deep infection of pathogenic fungi of the deep fungal infection. With the emergence of drug-resistant strains increasing, deep fungal infection pathogenic bacterium identification is extremely important. The traditional identification methods of fungal classification are analyzed according to the fungal morphology, physiological, biochemical characteristics, physiological and biochemical indexes, fungal culture and detection need special culture conditions and methods, and the majority of fungal grow slowly. These characteristics of fungi make the identification work of fungi great difficulties, the result of deep fungal infection of the pathogen identification of clinical significance was significantly lower. Molecular biology method is fast, accurate and gradually being used in clinical, DNA sequence analysis has become an important method to identificate the pathogenic fungi.Candida albicans is widely distributed in the nature atmosphere, usually it can invase the subcutaneous tissue, internal organs and mucous membrane of human body, and can cause systemic pathogenic fungus. Clinical common pathogenic fungi has Candida albicans, tropicalis Canida, Canida glabrata, Canida krusel and Canida parapsilosis, Among them, Candida albicans has became the most common found and most important pathogenic bacteria in clinic therapy.Gene family SAP of Candida albicans obtains at least nine members(SAP1 and sap9),which encodes a secreted aspartic acid protease Saps, aspartic acid proteases of Candida albicans involved in invasing tissue, which was recognized as aimportant pathogenic factors as pathogen infecting humans. The results show that when sap6 gene was knocked out, the viability of Candida albicans decreased and invasion of host cells decreased significantly, tissue damage could be reduced significantly.With the advantage of high efficacy, broad antimicrobial spectrum, high rate of absorption, strong security, azole antifungals are widely used for the prevention and treatment of candida infections. However, the long-term, widely and extensive use of the azole antifungals has gaven rise to blow up the drug-resistant strains, which has brought much trouble for the prevention and treatment of pulmonary candida infection.The mechanism that leads to the Candida resistant azole is complex and diverse; one of the most commonly reasons is the gene mutation or over expression of ERG11 gene, which is encoding drug target enzymes. The target enzyme of azole drugs in ERG11 gene encoding is 14-alpha demethylase(CYP51). ERG11 gene mutations cause the sequence of Ergllp’s amino acid change, further causes the spatial structure of the 14 alpha demethylase change, thus the binding between the enzyme and drug molecules couldn’t be completed or binding force is reduced, which makes fungi acquired drug resistance. The over expression of target enzyme of azoles can also make fungi acquired drug resistance. When resistant strains and sensitive strains were compared, we found that MIC value have increased in most target enzyme strains which has high m RNA.Comparative genomics is a discipline which based on the technology of genome mapping and sequencing, by comparison with the known gene and genome structure, thus uncover gene functions, expression mechanism and the evolution of species. By the comparison of ERG11 gene sequencing from the result of drug resistance of Candida albicans and standard Candida albicans, and compared with standard of Candida albicans ERG11 known genes, polymorphism differences existed in both gene sequences can be found and drug-resistance related gene might be located.In this thesis, ERG11 gene of Candida albicans is chosed as the study target,comparative genomics is adopted to operate a preliminary research on the relationship between ERG11 gene mutation and its drug resistance.Material and method57 qualified clinical strains of pulmonary fungal infection specimen who were treated in the department of respiration in the First Affiliated Hospital of Zhengzhou University were collected from 2013 March to 2014 March. The specimens were then inoculated onto the sand on Sabouraud medium, under 35 degree Celsius, 24~48h growth, during this time the spemicem grew into colony. Then, single colonies were selected and inoculated into CHROMagar Candida color culture medium. The colinies were identified according to the difference of the colony color, Merieux VITEK-32 automatic bacterial analyzing system of YBC card would be used to identify the bacteria if necessary.(Strains from the same position of the same person were excluded).ATBFugus3 test strip was used for drug sensitive test of 57 strains of Candida under standard operating procedures. The strains were cultured in aerobic conditions at 35 degree Celsius for 24 hours. At last, the interpretation result of drug sensitivity was assured and record. The Azole antifungals selected include fluconazole, voriconazole and itraconazole.We selected conserved sequences SAP6 of Candida albicans as aim gene to be detected, and designed the C.albicans SAP6 gene PCR primers by Primer5.0 software design according to Candida albicans sap6 gene sequence in Gen Bank, and the PCR products were purified and sequenced after the amplificating of the target gene, then contrsting with the SAP6 sequencing. When the PCR products sequenced result is same with the sequences SAP6, we think it is Candida albicans.The DNA of all tested Candida albicans was extracted. Based on the gene sequence of Candida albicans published by Gen Bank, PCR primers of Candida albicans ERG11 gene was designed by software Primer5.0. After amplification, the PCR products were purified and sequenced. Using Blast analysis software, the standard sequence of X13296 gene sequence in the sequencing results and the Gen Bank database published were compared and analyzed, to determine whether a gene mutation exist or not.ResultFirst, Among the 57 identified strains; the majority of the tested strains was Candida albicans, with 32 strains, accounts for 56.1%; followed by Candida glabrata, with 12 strains, accounting for 21.1%, Candida tropicalis, with 8 strains, accounting for 14%; Candida krusei, with only 3 strains, accounts for 5.3%; the others, with 2 strains, accounts for 3.5%.Second, Vitro drug sensitivity results of 57 strains of Candida albicans are as follows: among the 32 strains of Candida albicans, fluconazole, 4 strains, itraconazole, 7 strains, Candida glabrata voriconazole 2 strains; 12 strains, fluconazole resistant were 2 strain, itraconazole drug resistance of 3 strains, none of the strains of Candida tropicalis voriconazole resistance: 8 strains, 3 of the strains were resistant to fluconazole, itraconazole resistance 1 strains, 1 strains of voriconazole resistance; Candida krusei to fluconazole resistance of 3 strains, 1 strains, 2 of the strains were resistant to itraconazole, voriconazole drug resistance of 0 strains; 2 strains of other beads, no resistance.Third, The SAP6 gene of 32 isolates of Candida albicans was amplified and sequenced, and SAP6 genes were detected in the Candida albicans.Fourth,32 strains of Candida albicans ERG11 gene were sequenced, compared with the standard sequence of X13296, a total of 37 base pair mutations, including 11 missense mutations a. The sensitive strains 3 strains appear missense mutations, 9 strains of resistant strains exist 1~5 missense mutation. The tested results revealed that Candida albicans in ERG11 gene missense mutations and amino acid changes, including missense mutations have sensitive strains T945 A, G1309 A, A530 C, the corresponding amino acid mutation for D116 E, V437 I, K128 T, T495 A, A530 C, T541 C resistant strains, G622 A, G979 A, A945 C, G1309 A, G1496 A, G1728 T, D116 E, K128 T and the corresponding amino acid mutation Y132 H, V159 I, D278 N, E226 D, V437 I, G450 E, M527 I, respectly. ConclusionBy the sequencing of ERG11 gene in Candida albicans, it revealed that most of the Candida albicans which is resistance to antifungal drugs have the ERG11 gene mutation. Mutation of gene ERG11 in Candida albicans has led the changes in amino acid V437 I, D116 E and K128 T, it’s unrelated with Candida albicans’ resistance to three antifungal agents; the changes in amino acid D226 E, G450 E and Y132 H are related with Candida albicans’ resistance to three antifungal agents; while the relationship between changes in amino acid V159 I, D278 N, M527 I and Candida albicans’ resistance to three antifungal agents still needs further research. Multi-point missense mutation could produce synergies, which lead to the strengthen of resistance or cross resistance. Meaningful amino acid changes haven’t been searched in many Candida albicans which were resistant to three antifungal agents, which implies that other mechanisms take part in its resistance.